Field of the Invention
Measurement of total Glutathione in blood and other body fluids and tissues must be accurate in order to have diagnostic significance or the ability to participate in drug metabolite screening.
Description of Related Art
Previously, glutathione deficiency was examined as a possible prognostic for survival in Acquired Immunodeficiency Syndrome (AIDS), as disclosed for example in U.S. Pat. No. 5,843,785. In that patent, general glutathione levels of whole blood and/or T-cells of HIV positive patients were determined (directly or indirectly) as an indication of the likely period of survival, as well as the need for agents for enhancing the glutathione levels. But the determination made was not accurate enough to make any kind of correlation with survival expectancy of HIV positive patients. Moreover, it was also mentioned “referring to glutathione levels, it will be understood that it is the value determined by an assay which can be used for comparisons, but does not provide an absolute value”. Therefore since the glutathione determinations were not accurate and absolute, the stated hypothesis was not valid enough to corroborate probability of survival in HIV positive patients. The probability of survival expectancy of HIV positive patients does not depend on one particular biomolecule measurement such as glutathione, but on several other conditions associated with the etiology of the disease. The methods both (HPLC and FACS) used for the determination glutathione levels are not robust enough to address the problems associated with quantification like reduced glutathione oxidation, and conjugates formation.
Similarly, in Pastore, G. Federici, E. Bertini, F. Piemonte, “Analysis of glutathione: implication in redox and detoxification,” Clinical Chim Acta 333 (2003) 19-39, blood concentrations of reduced glutathione (GSH) in various pathologic conditions, and the statistical significance of the purported validation one or more sub-species, are indicated as being diagnostically significant. Unfortunately, most if not all of the asserted data are not accurate, which presents a significant problem for physicians in assessing and treating patients. In many cases the imprecision of these GSH and GSSG numbers overlap as they are not compared with confidence intervals but with means which are not statistically different.
United States Published Patent Application No. US2011/0144205 A1 hypothesized that glutathione is a key biomarker for heart failure asymptomatic patients, stating that “inflammation and oxidative stress are key components of in the pathophysiology and progression of heart failure and are strongly associated with the disease severity.” Chemically, oxidative stress is associated with increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses. Therefore, these antioxidant defenses can be not only glutathione but also can be vitamin E, vitamin C, enzymes like superoxide dismutase, metallothionein, etc. The method for quantification of glutathione mentioned is not accurate and precise enough to correct for glutathione oxidation and conjugate formations.
In “Methods for the Quantitation of Oxidized Glutathione,” U.S. Pat. No. 6,235,495 B1, the method used for quantification of oxidized glutathione based on the assay developed by Tietze, which is based on spectrophotometric determination of the reaction of the Ellman's reagent with Glutathione disulfide. The observation is made by assessing the color at a particular absorbance. The invention mentioned in this patent is the use of 1-methyl-2-vinylpyridinium trifluromethanesulfonate (M2VP) to prevent the conversion of GSH to GSSG without interfering with glutathione reductase in the biological sample. However, this method is not accurate and precise enough to make since clinical and therapeutic decisions, it is based on the color change at particular absorbance and any other disulfide present in the biological sample can interfere with the process and can lead to false positive result.
The invention in the published U.S. Patent Application No. US2009/0029409 involves determination of oxidative stress biomarker, which can be varied by perturbation of glutathione levels in the blood. The oxidative stress biomarker candidate identified is ophthalmic acid, but the mechanism as to how glutathione is affected has not been established clearly. Although reduced glutathione is present in low concentrations and prone to oxidation in blood, it still can be quantified by using NEM in the sample preparation which minimizes the conversion of reduced glutathione to oxidized glutathione. The normal range of ophthalmic acid present in the blood has not been mentioned and whether its production is varied by any other biomolecule other glutathione has not been discussed.
Zhenying Yan and coinventors have described, in two US patents, 2005/US0287623 and 2011/US788186, a method for detecting reactive metabolites by isotope trapping with a mass spectrometer for the purpose of toxicological assessment. In both Yan patents, the European patent by Michael J. Avery (EP, 1,150,120, October 2001) has been provided as a prior art that involves incubating a test compound with a microsomal drug metabolizing enzyme system in the presence of a form of glutathione and then, detecting certain glutathione adducts formed therefrom using tandem mass spectrometry. Yan points out that Avery method will identify reactive metabolites as well as non-reactive components (including both unreactive metabolites and components of the reaction mixture) formed as a result of common response in mass spectrometry detection, thus resulting in false positives. In the 2005-Yan patent, a method of detecting reactive metabolites of a drug candidate whereby the drug candidate was mixed with a non-labeled trapping agent, an isotopically labeled trapping agent and an enzyme. Purportedly improving on the 2005-patent, Yan and Norman D Huebert, U.S. Pat. No. 7,884,186 B2, disclosed in the 2011 text that the 2005-patent detects only “soft” metabolites, but does not simultaneously detect both “hard” and “soft” reactive metabolites. In the 2011 Yan specification, the “soft” metabolite is described as any electrophilic metabolite which comprise at least one substituent group which readily reacts with soft electrophiles, such as the sulfhydryl group in cysteine and the —SH group on the compound of the formula. Disclosed examples of soft metabolites were given as quinones, quinone imines, immunoquinone, methids, epoxides, arene oxides and nitrenium ions. The same patent described a hard metabolite as an electrophilic metabolite which comprises at least one substitute group which readily reacts with the —(CH2)4—NH2 of the compound of the formula. The disclosed example of such substituent group was given as aldehydes. Additionally, the same patent claims that its isotope trapping technique was capable of eliminating false positives using mass spectrometric pattern recognition. There is no indisputable evidence in either of the Yan patents that would support the claims associated with the “elimination of false positives.” Without standard reference materials, a mathematical basis and statistical support, such claims would not meet the criteria established for mission critical applications such as those established in the clinical, diagnostic and homeland security fields.
In the above exemplary prior art, the field attempts to identify various glutathione adducts and conjugates has therefore been fraught with fundamental problems. A need remains, therefore, for an accurate method of measuring and calculating tGSH and specific related compounds and conjugates in a patient's blood, fluid or tissue sample and the concomitant diagnostic method of interpreting the measurements and ratios for diagnostic and prognostic purposes. A need also remains for improved accuracy in situ toxicological drug screening of biologic drugs involving GSH, GSH species, GSH conjugates, and GSH metabolites.